Anthrax toxin, a major virulence factor of Bacillus anthracis, consists of the cellular binding moiety protective antigen (PA) and the enzymatic moieties lethal factor (LF) and edema factor (EF). To intoxicate host organisms, PA binds to its cellular receptors TEM8 and CMG2 and is proteolytically activated by the ubiquitously expressed cell surface furin protease, resulting in the formation of active PA heptamer, which in turn translocates LF and EF into the cytosol of cells. LF cleaves several MEKs, thereby inactivating the ERK, p38, and JUNK MAPK pathways. EF is an adenylate cyclase that generates abnormally high concentrations of cAMP. PA is a key virulence determinant of anthrax disease, and antibodies to PA protect against infection. Thus, PA is the focus of existing and new vaccines. Our group is continuing its long-term effort to improve methods for producing PA and PA-based vaccines. Avirulent strains of B. anthracis are being engineered to be sporulation deficient and to lack extracellular proteases. Proteins secreted from such strains are more easily purified with higher yields and with increased stability. During 2010, we evaluated additional B. anthracis strains having multiple extracellular proteases deleted. More details about these strains are provided in the report for project AI001030-03. In addition, we recognized the value of adding additional calcium ions to the growth medium. PA contains two tightly bound calcium ions, and it appears that adding calcium improves the folding of PA as it is secreted from the bacteria. The added calcium causes yields to increase 2-fold over previous levels, reaching nearly 100 mg per liter. Another approach to immunological defense against anthrax uses passive administration of monoclonal antibodies. We are continuing collaborative work with Dr. Purcell (LID, NIAID) on chimpanzee/human monoclonal antibodies (mAbs) to various anthrax antigens. In parallel work, we are developing new mouse mAbs against the anthrax toxin enzymatic moieties, EF and LF, to better understand immune responses to the toxins, and to identify mechanisms by which antibodies neutralize the toxins. Thus, during 2010 we prepared a new set of mAbs to EF and showed that they react with different epitopes and have several mechanisms of action. These mAbs were shown to be useful in detection and measurement of EF in the blood of infected animals. During 2010 we also arranged to obtain a set of incompletely characterized mAbs to LF. Full characterization of these mAbs is expected to identify reagents of value in diagnostics and in the characterization of toxin action. One area of work in this project seeks to use modified anthrax toxins to target cancer. We previously found that the toxicity of anthrax toxin can be redirected to cancers by changing PAs furin specificity to cancer-selective protease specificities. Therefore, we constructed uPA (urokinase type plasminogen activator) and MMP (matrix metalloproteinase) activated anthrax lethal toxins. uPA and MMPs are proteases that are overproduced by cancer tissues and required for invasive growth, serving as potential molecular targets for cancer treatments. We previously found that the MMP-activated LeTx has a potent anti-cancer activity due to its targeting of tumor vasculature. In 2010, collaborating with Dr. Arthur Frankel (Cancer Research Institute, Scott and White Memorial Hospital, Temple, Texas), we found that the MMP-activated LeTx is also very potent in treating human anaplastic thyroid carcinoma (ATC) in an orthotopic mouse model. We showed that the MMP-activated LeTx inhibits orthotopic ATC xenograft progression via reduced endothelial cell recruitment and subsequent tumor vascularization. This in turn translates to an improved long-term survival in mouse models that is comparable with that produced by the multikinase inhibitor sorafenib. The results also indicate that therapy with the MMP-activated LeTx is extremely effective against advanced cancers with well-established vascular networks. The results demonstrate that MMP-activated LeTx-mediated endothelial cell targeting is the primary in vivo antitumor mechanism of this novel toxin, suggesting its potential usefulness in the clinical management of many different types of solid tumors. In the year of 2010, we also developed a "humanized uPA" mouse strain. In this strain, termed Plau(GFDhu/GFDhu), we introduced four amino acid substitutions into the growth factor domain of uPA, resulting in a mutated uPA that can bind only to human uPAR but not to the endogenous mouse uPAR. In this strain, the interaction between endogenous uPA and uPAR is selectively abrogated, while other functions of both the uPA protease and its receptor are retained. Elucidation of the specific functions of the uPA-uPAR interaction in vivo has been difficult because uPA has important physiological functions that are independent of binding to uPAR and because uPAR engages multiple ligands. It was in part to resolve this problem that we, in collaboration with Dr. Thomas Bugge (NIDCR), generated this humanized uPA mouse strain using a gene targeting approach. Analysis of the Plau(GFDhu/GFDhu) mice revealed an unanticipated role of the uPA-uPAR interaction in suppressing inflammation secondary to fibrin deposition. In contrast, leukocyte recruitment and tissue regeneration were unaffected by the loss of uPA binding to uPAR. This study identifies a principal in vivo role of the uPA-uPAR interaction as being the promotion of the cell-associated fibrinolysis critical for suppression of fibrin accumulation and fibrin-associated inflammation. This mouse provides a valuable model for further exploration of the multifunctional uPAR receptor. In addition, this mouse will have value in evaluating the anti-cancer efficacy of uPA-activated anthrax toxins. We previously showed that the uPA-activated anthrax toxin has a potent anti-cancer efficacy in two syngeneic mouse cancer models. In these models uPA, which often is secreted by host-derived tumor stromal cells, binds to uPAR on cancer cells and activates the cell-associated uPA-activated anthrax toxin, resulting in killing of the cancer cells. However, because mouse uPA cannot bind human uPAR, this toxin is not expected to be active against human tumor xenografts in mouse models. The new mouse described here will allow such efficacy tests. This laboratory has for many years done mutagenic and somatic cell genetic analyses to identify host genes involved in the actions of bacterial protein toxins. We have previously used retroviral insertional mutagenesis to isolate anthrax toxin receptor-deficient CHO cell mutants and mutants defective in diphthamide modification on eukaryotic elongation factor-2 (eEF-2). Because diphthamide is the target of bacterial ADP-ribosylating toxins (such as Pseudomonas exotoxin A), the diphthamide-deficient CHO cell mutants are completely resistant to these bacterial toxins. In 2010, we completed the analysis of an additional CHO cell mutant obtained in the earlier selections. CHO PR3228 belongs to another class of CHO cell mutants that are resistant to the bacterial ADP-ribosylating toxins. We have now shown that these cells have a mutation in one allele of the eEF-2 gene. This mutation, Gly717Arg, is close to His715, the residue that is modified to become diphthamide. This Arg substitution prevents diphthamide biosynthesis at His715, rendering the mutated eEF-2 non-responsive to ADP-ribosylating toxins, while having no apparent effect on its function in protein synthesis. CHO PR328 cells are heterozygous, having one mutant eEF-2 allele which allows the cells to survive even in the presence of ADP-ribosylating toxins, indicating that 50% of active eEF-2 is sufficient to support long term cell survival.